The present invention relates to a reagent useful for analysis of triglyceride and an analysis method of triglyceride and an analysis method using the same.
More specifically, the present invention relates to a reagent comprising lipases and monoglyceride lipases, which is useful for the rapid quantitative analysis of triglyceride, particularly those contained in blood serum, and an improved method for the analysis of triglycerides using the same.
The triglyceride contained in samples, particularly those such as body fluids, has been analyzed in such a manner that the triglyceride is hydrolyzed usually using lipases ultimately up to glycerol and fatty acid through diglyceride and successive monoglyceride, and then the glycerol or fatty acid liberated from the triglyceride is assayed by a method taking advantage of an enzymatic or chemical reaction. The assay is conducted, for example, in a simple manner, by the use of electrodes or indicators.
In order to perform an effective analysis of triglyceride contained in blood serum, proteolytic enzymes such as proteases and lipoprotein lipases have been used for liberation of triglyceride from bound proteins, followed by analyzing the triglyceride. Japanese Patent Publication No. 9518/1979 (Japanese Patent Application No. 114493/1978) proposes to use both lipases and proteases. In addition, in order to promote the hydrolysis reaction of triglycerides to form glycerol, a combination of various lipases having different properties from each other, and an addition of surface active agents or chemical agents to the sample solution have been proposed. There are, for example, hydrolysis using a combination of lipases having different properties from each other (Japanese Patent Laid-Open No. 25694/1977, Japanese Patent Publication No. 29/1981 and Japanese Patent Publication No. 28276/1982), hydrolysis using a combination of lipases and cholesterol esterases (Japanese Patent Publication No. 46799/1981), hydrolysis using a combination of lipases and surface active agents (Japanese Patent Publication No. 39158/1982), hydrolysis using a combination of lipases, surface active agents, and phenol or aniline derivaties (Japanese Patent Publication No. 5677/1983), hydrolysis using a combination of lipases, carboxyesterase originated from pig livers and alkali metal or alkaline earth metal alkylsulfates (Japanese Patent Laid-Open No. 64495/1974), and hydrolysis using lipases, pancreatic lipases and salts of bile acid (Japanese Patent Laid-Open No. 11987/1977).
As described above, the conventional analytical methods have been carried out by separating triglyceride from bound proteins, and promoting the hydrolysis reaction of triglyceride to produce glycerol. However, hydrolysis rate is not so speedy that a considerable period of time is needed for the analysis, since almost all lipases used in these methods are capable of hydrolyzing the triglyceride up to glycerol, but they are not active enough on the .beta.-monoglyceride produced by the hydrolysis of triglyceride, i.e., have low activity in hydrolyzing monoglyceride to glycerol. Therefore, it has been desired to accelerate the hydrolysis reaction of the monoglyceride to produce glycerol and shorten the period of time for analysis.
The present inventors succeeded in isolating a monoglyceride lipase-producing microorganism belonging to Bacillus stearothermophilus H-165 strain, which is able to catalyze the hydrolysis reaction from monoglyceride to glycerol, from the soil around the hot spring of the spa of Kirishima, Kagoshima-ken, Japan, and a monoglyceride lipase is obtained by the cultivation thereof (Japanese Patent Application No. 80299/1987). The inventors have also found that the resulting monoglyceride lipase is capable of acting on .alpha.- or .beta.-monoglyceride extremely strongly unlike the conventional lipases which act weakly on the monoglyceride. Moreover, it has been found that the monoglyceride lipase has an optimum pH value of around 5, an isoelectric point of 4.6 and a molecular weight of as low as 27,000, shows a maximum activity at an optimum temperature of 75.degree. C., and is excellent in the heat resistance so that it is hardly inactivated at 70.degree. C. and holds the activity by 20 % even after the treatment at 90.degree. C.
The present inventors have further studied extensively to find that the combined use of the monoglyceride lipase and lipases is able to shorten the period of time required for the analysis of the triglyceride contained in a sample solution, because the monoglyceride lipase specifically strongly acts on the monoglyceride formed from the triglyceride through diglyceride by the action of lipase, resulting in the acceleration of the hydrolysis reaction from the triglyceride to glycerol.